Bone graft

ABSTRACT

The present invention relates to a novel bone graft and methods for producing the graft. The bone graft can be used for surgical, plastic and/or cosmetic bone replacement for a patient in need thereof. The bone graft is made of a scaffold or matrix of sheet material having a 3-dimensional pattern of a continuous network of voids and/or indentations for enhancing new bone growth.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/303,811 filed onNov. 23, 2011.

FIELD OF THE INVENTION

The present invention relates to a novel bone graft and methods forproducing said graft. Said bone graft can be used for surgical, plasticand/or cosmetic bone replacement for a patient in need thereof, or usedas a means of delivering medicaments and defining marrow constituents.

BACKGROUND OF THE INVENTION

Orthopaedic surgeons have been applying the principles of tissueengineering for years, while transplanting and shifting matrices withinpatients to promote regenerative potential. The advent of new technologynow offers even greater promise and brings unbridled enthusiasm thatfull regenerative potential of tissue and whole organ systems can beachieved in the near future. While soft tissue repair can be managed byachieving scar tissue replacement, such outcome in most orthopaedicapplications and indications would be insufficient. Bone requires atissue-specific composition to be attendant to function for skeletalsupport. The forming of collagenous material alone, even ifvascularized, will fail to meet the biophysical demands of repetitiveskeletal loading and be inadequate.

Implicit in the goals of repairing bone are to achieve restitution ofspace, mechanical solidarity, and functional continuity. Often thebiological signals do not provide sufficient stimulus to attain a fullrepair. Orthopaedic interventions to alleviate fracture non-union,pseudarthrosis, and scoliosis; bone defects due to congenital ordevelopmental anomalies, infection, malignancy, or trauma often requirebone grafting to augment the process of bone healing. The therapeuticgoal of graft material is to omit compliance features such as straintolerance, reduced stiffness, and attenuated strength, and insteadpromote primary, or membranous-type bone formation within the physicalapproximation of graft material. In order to achieve this, three basiccomponents are required: osteoprogenitor cells, osteoinductive factors,and an osteoconductive matrix or scaffold.

Autologous cancellous bone remains to date the most effective graftmaterial, where osteoinductivity, osteoconductivity, and a rich sourceof cells endow the material with not only biological activity but adegree of immunologic transparency as well. Because of complications andshortcomings associated with autogenous grafting that include limitedquantity, donor-site morbidity, and more recently cost consideration(St. John T. A., et al., Am. Journal of Orthopedics; 32:18-23, 2003),numerous alternative graft materials have been developed for orthopaedicapplications. All references as cited herein are incorporated in theirentireties for the purposes of the present invention.

Allograft bone is used extensively as a material bridge toosseo-integration, acknowledged as a substitute for the general shortsupply of autograft. Primary uses to date have been in spine, althoughboth trauma and plastics account for a growing market. The market offersseveral options, the most valuable being the machined components forsurgical implantation. Over 675,000 procedures set the demand forallograft annually, with a projected market growth set at 19%.

Available grafting substitutes include cancellous and cortical allograftbone ceramics such as sintered coralline matrices, hydroxyapatite andtri-calcium phosphate, demineralised bone matrix, bone marrow, compositepolymer grafts, and recently recombinant cytokines with collagencarriers. Complications include availability, cost, variablebioabsorption, brittleness, immune stimulation, and regulatory hurdles.

The shape of the biomaterial template is critical to the success ofmanufacturing. A central tenet of biomineralization is that nucleation,growth, morphology and aggregation of the inorganic crystals of bone areregulated by organized assemblies of organic macromolecules. The closespatial relationship of hydroxyapatite crystals with Type I collagenfibrils in the early stage of bone mineralization is a relevant example.It is equally evident that combining hydroxyapatite with protein doesnot render the macroscopic form of bone nor impart its characteristicproperties. Unlike fabricated materials that can be developed fromcomponents with predictable properties (Olson G. B., Science; 277:1237-1242, 1997), biological systems control desired properties byutilizing an intrinsic rationale that discriminates essential fromnon-essential factors. Living organisms avoid the geometric frustrationof randomness by segregating structures that resonate function.

Future envisaged bio-engineering strategies will combine severalfavourable properties of the current items in an effort to achievehybrid materials that support tissue differentiation without shieldingcapacity for integrated modelling.

Ideally, new materials will provide tissue compatibility and minimizepatient morbidity.

Although bone can appear de novo, it more often develops from accretionon a scaffold of matrix that contains appropriate vascular andcompositional arrangement. As such, both 2-dimensional and 3-dimensionalpatterns have been shown to enhance osteoconductivity (Liao H., et al.,Biomaterials 24: 649-54, 2003). Bone has significantly more matrix thancells, and cell regulation through anchorage dependent mechanisms is anestablished premise (Clover J. and Dodds R. A., J. Cell Sci; 103:267-271, 1994; Ingber D. E., Int. Rev. Cytol.; 150: 173-224, 1994;Meazzini M C et al., J. Orthop. Res.; 16: 170-180, 1998). Compensatorymechanisms for changing sensitivity to mechanical stimulation have beenshown to undergo adaptive or kinetic regulation, likely tied, directlyto osteoblast attachment to immobilized molecules in the extracellularmatrix (ECM). ECM molecules promote cell spreading by resisting celltension, thereby promoting structural rearrangements within thecytoskeleton (Ingber D. E., Annu. Rev. Physiol.; 59: 575-599, 1997).Several lines of evidence suggest that tension or mechanical stretchexerts a direct positive effect on bone cells and bone celldifferentiation through: 1.) activation of phospholipase A2, 2.) releaseof arachidonic acid; 3.) increased prostaglandin E synthesis, 4.)augmented cyclic adenosine monophosphate (cAMP) production; and 5.) andexpression of the bone associated transcription factor CBFA-1(Bindermann I., et al., Calcif Tissue Int.; 42: 261-266, 1988; SomjenD., et al., Biocim. Biophys. Acta; 627: 91-100, 1980; Yeh C. K. andRodan G. A., Calcif Tissue Int.; 36: S82-85, 1984; Nikolovski J., etal., FASEB J.; 17: 455-7, 2003). It has long been recognized that asustained increase in the cellular level of cAMP constitutes agrowth-promoting signal (Rozengurt E., et al., J. Cell Biol.; 78:4392-4396, 1981), and that prostaglandins directly affect a change incell shape and increase intercellular gap junctions (Shen V., et al., J.Bone Miner Res.; 1: 2443-249, 1986). Without a capacity for attachmentand spreading, cells undergo apoptosis, or programmed cell death (ChenC. S., et al., Science; 276: 1425-1428, 1997; Edmondson A. C., Bosten,1987).

Bone withstands compressive loading by efficient distribution ofinternal tensile forces. Bone cells do however adhere to structures thatcan resist compression in order to spread, engaging osteoblastattachment, mineralization, and bone matrix organization as linkedprocesses. Even though deformation at the tissue level might beevaluated as an ability to resist compression, force along individualtrabeculae reflects an ordinate of new tension. Under normal cycles ofdevelopment, increased mass conveys a progressive stimulus of tension tocells, gravity imposing a unidirectional vector to terrestrial life.

A sudden reduction in gravity imposes serious consequence to theskeleton. As shown by studies of astronauts, marked skeletal changes inthe weight-bearing skeleton including a reduction in both cortical andtrabecular bone formation (Jee W. S. S., et al., Am. J. Physiol.; 244:R310-R314, 1983), alteration in mineralization patterns (Zerath E., etal., J. Appl. Physiol.; 81: 194-200, 1996), and disorganization ofcollagen and non-collagenous protein metabolism (Backup P. K., et al.,Am. J. Physiol.; 266: E567-E573, 1994) have been associated withmicrogravity. Each month of spaceflight results in a 1-2% reduction ofbone mineral density that has been linked to down-regulated PTH and1,25-dihydroxyvitamin D3 production (Holick M F, Bone; 22: 105S-111S,1998). Indices from cosmonauts aboard Euromir 95 account bone atrophy toboth a reduction in bone formation and increased resorption. PTHdecreased (48%), as did bone alkaline phosphatase, osteocalcin, andtype-I collagen propeptide. At the same time bound and freedeoxypyridinoline and pro-collagen telopeptide increased(Caillot-Augusseas A., Lafage-Proust M H et al., Clin. Chem.; 44:578-585, 1998). The chords of information establish a role formicrogravity in uncoupling bone formation and enhancing resorption.

If exposure to microgravity demonstrates physiologic responses thatmirror a reduction in trabecular tension, then would reciprocity offunction be expected in bone that is modelled under microgravity andthen exposed to normal gravitational force? Prolonged weightlessness, asexperienced in space flight effectively unloads the skeleton, relaxingtension on the trabeculae. In this manner, osteoblast physiologypreferably is altered due to attachment perturbations. Conversely, abioscaffold modelled in the form of tissue that has developed undermicrogravity, will experience an enhanced tensile loading sensation onindividual trabeculae.

In view of the above it is therefore the object of the present inventionis to provide a scaffold that preferably is not only structurallyenhancing but at the same time inductively optimum for bone formation.This graft should be designed to stimulate cell differentiation and boneregeneration, and to be utilized as an orthotopic alternative to tissuetransplantation.

SUMMARY OF THE INVENTION

The present invention overcomes the disadvantages of the art asdescribed above and, in a first aspect of the invention, provides asheet material for a bone matrix scaffold, comprising a surface patternthat is enhancing osteo-conductivity, wherein the surface pattern is a3-dimensional substantially continuous network having voids, or surfaceindentations that may or may not penetrate the sheet.

In the context of the present invention, the term “enhancingosteo-conductivity” shall mean the ability to change surface roughness,engage surface energy, promote mimetic patterns that resolve randomnessand integrate form and functional regenerative clues, that might alsoinclude appropriate bioactive regenerative adjuvants such as proteins,peptides, charge, shape, size, allometry, isotropy, anisotropy, ornon-biologic energies such as electric field, vibrational energy,gravitational variation, pressure wave, or material compositions thatwould enhance tissue attachment and reduce proliferative response.

In a preferred sheet material according to the present invention, thesheet has a thickness of about 150 micron. The voids can preferably havea medium width of about 10 microns, and at least 10% of said voids havea fractal dimension of at least 3. The sheet can be thicker and thevalue of the invention is the surface regularity. In purpose, the sheetcould be between 150 micron and 2 mm in thickness, with surfaceindentations ranging from 10 micron to 150 micron. Neither side of thesheet need be mirrored (as in image), and depth of indentations can varyby location to best accommodate geometric form.

Even more preferred is a sheet material according to the presentinvention, wherein the sheet is made of a biological cellular, cellularas a source of its production, or in containing cell fragments topromote regenerative cues, material hereinafter referred to as abiomaterial, preferably consisting essentially of collagen. Herein,“essentially” shall mean that more than 95% of the initial material ofthe structure, preferably more than 98%. Among the wide variety ofsubstrates that could be used as a basis for cultured allograft,collagen uniquely supports several desirable features. First, collagenis a principal component of bone, contributing more than 90% to theorganic composition. Second, in studies demonstrating osteoinductivecapacity of demineralised bone matrix, 60% of the stimulatory activityis derived from the collagen-associated component (Sampath T. K. andReddi A. H., Biochem Biophys. Res. Commun; 119: 949-54, 1984). Third,human collagen has been approved for injection and demonstrates minimalimmunoreactivity. Nevertheless, the use of other collagens and othersources of collagen (e.g. chicken, etc.) are envisaged as well. Anotherembodiment of the material would include ceramics, non-organicmaterials, electrospun nanofilaments, and nanoceramic crystals thatmight be either polararized, or aligned by their fabrication to amicroelectrical preference.

In another embodiment of the present invention, the material of thesheet is essentially biodegradable, bioerodable and/or bioresorbable.“Biodegradable, bioerodable and/or bioresorbable and/or bioinstructive”in the context of the present invention shall mean capable of beingdecomposed by organisms, in particular the human body. Furthermore, thesheet material shall be able to decay naturally and harmlessly intoparts that are resorbable by organisms, in particular the human body.“Essentially” shall mean that more than 95% of the initial material ofthe structure is decomposed, preferably more than 98%.

The invention duplicates the architecture of under-modelled cancellousbone, guided by the idea that a material later populated with bone cellswill more quickly respond to the mechanicals and biological roles ofbone with subsequent loading. Because cancellous bone is a porousstructure, its mechanical properties are dependent upon the distributionand arrangement of its structural elements, or trabeculae. Consideringthree-dimensional architecture to be critical to the mechanicalintegrity of trabecular bone, the applicant established the morphometryof under-loaded marine mammal tissue (Ganey T. M., et al., 44^(th)Annual Orthopaedic Research Society, 1998). The rationale for thepresent approach is based on the observation that prenatal cancellousbone in humans has unique potential for rapid post-natal modelling(Ogden J. A., Springer Verlag, 1999), and that cell-culture studiesperformed during orbital space flight demonstrate significant osteoblaststimulation upon return to increased gravitational field (Harris S. A.,et al., Bone 20(4) 26: 325-31, 2000). In the case of sea mammals,separate environment buoyancy suppresses loading variation, resulting inminimal secondary bone formation and modelling. Whale bone retains aprimary trabecular structure and does not remodel according to standardparameters of mechanical adaptation. Trabecular morphology and osteocytenumber are similar among commonly oriented blocks, while significantdifferences can be demonstrated between tissue sections studied inplanes perpendicular to the axial length of bones.

TABLE 1 BIOPSY BV/TV BS/BV TbTh TbSp TbN Ost# Cross 17.71 14.98 135.16631.70 1.33 230/mm² Long 24.64 8.67 231.05 710.09 1.06 150/mm²BV/TV—Bone Volume/Tissue Volume; BS/BV—Bone Surface/Bone Volume;TbTh—Trabecular Thickness = μm; TbN—Trabecular Number; Ost#—osteocytecells per mm²

Bone examined in longitudinal dimension demonstrated greater trabecularseparation, thicker trabeculae, yet because of the lesser number oftrabecula, still structured less bone surface per volume of tissue.Although bone surface to bone volume, trabecular thickness, andtrabecular number followed predicted allometric extrapolation, reflectedin greater trabecular separation and reduced trabecular number. It isthis separation and thickness that provides a basis for bio-reactor cellculture and offers the chance to manufacture bone as claimed in thisapplication.

Reddi and Huggins demonstrated the effect of open-ended vs. close-endedtubes on cell differentiation, showing bone rather than cartilage as therespective outcome (Reddi A. H. and Huggins C. B., Proc. Soc. Exp. Biol.Med.; 143: 634-637, 1973), and further confirming the importance ofparticle size in the activity of demineralised bone matrix (Sampath T.K. and Reddi A. H., J. Cell. Biol.; 98: 2192-2197, 1984). Still otherstudies have shown the importance of geometry when considering theeffective integration of an implant, particularly the porosity andinterconnections of the matrix (Borden M., et al., Biomaterials; 24:597-609, 2003). To assure that the type-I collagen matrix will maintainits structure during the cell culture process and avoid the toxicity ofother cross-linking agents, a non-aldehyde cross-linking processpreferably is utilized to stabilize the construct (Koob T. J., et al.,J. Biomed. Mat. Res.; 56: 31-39, 2001; Koob T. J., et al., J. Biomed.Mat. Res.; 56: 40-48, 2001). Previous work by Koob and Hernandezdetermined that pepsin-solubilized type-I collagen fibers can bepolymerized with nordihydroguaiaretic acid (NDGA), and plant compoundwith antioxidant properties. In tendon, the process produces fibers withmaterial properties in uniaxial tensile tests to failure that arecomparable to native tendon (Koob T. J. and Hernandez D. J.,Biomaterials; 23: 203-212, 2002).

The role of scaffold material as a delivery vehicle for osteogenicmaterials is best understood in the context of bridging critical-sizedefects. Among the key roles of the sheet material and scaffold is tooptimize the location and release of the biogenic factors whileinsulating the space from soft-tissue encroachment. Methods for designand development of collagen-based bone prostheses have focused oncross-lining agents in order to improve the tensile properties of thecollagenous constructs and to reduce the potential inflammatory responseto foreign materials. Optimizing the geometry of the prosthesis is acritical engineering strategy for assuring matrix deposition, andcross-linking of the collagen matrix (sheet material) supports theinterim scaffold to effect cell attachment.

The concept for manufactured graft, in particular bone allograft,depends on a capacity to achieve reproducible design in a geometricscaffold. Such scaffolds will support osteoblast attachment and permitbone-specific matrix production. In light of anticipated rulings by theFDA for greater control of tissue products for transplantation,developing alternative materials with comparable osteoinductivity seemsappropriate. Several features combine to make this invention unique;first, the bone model for the sheet material and scaffold architectureapproximates an under-modelled mammal, using increased porosity toaccelerate ingrowth; second, a unique cross-linking methodology reducesthe bioabsorption rate of polymers (e.g. human collagen) and effects amechanically competent scaffold; and third, osteoblasts can be used todeposit a bone matrix onto the scaffold that will make itosteo-inductive.

The controlled process according to the present invention is intended totake advantage of previous regulatory considerations of human collagenas a device. FDA approval for human collagen in combination withincipient material is not unprecedented and will lower the threshold formarketability.

Another aspect of the present invention is thus directed to a method forproducing a sheet material for bone grafts, comprising the steps of a)providing a rectangular sheet of suitable biomaterial, and b)introducing a surface pattern into said suitable biomaterial, whereinsaid surface pattern is enhancing the osteo-conductivity of saidbiomaterial and is a 3-dimensional substantially continuous networkhaving voids. Preferably said method produces a sheet that has athickness of about 150 micron. Nevertheless, thinner sheets can beproduced that are to be used in laminate structures, these thinnersheets are herein designated as “thin wafers”. A preferred thickness ofsuch wafers preferably is between 150-300 nm and the individual fibersattending the sheet fabrication preferably range from 50-150 nm. Theseindividual sheets will be by design variable in porosity but accountableto isotropic layering so that fundamental randomness to directed loadingis retained.

More preferred is a method according to the present invention, whereinsaid biomaterial is collagen, in particular type-I collagen, derivedfrom suitable sources as indicated above.

Generally, the surface pattern of the inventive sheet material can beprovided to said sheet material by all suitable techniques that allowfor the introduction of a surface pattern having the correct dimensions.Preferred is a method according to the present invention, wherein saidsurface pattern is introduced by etching and/or embossing and/ormicroprinting the surface or surfaces of the biomaterial. The surfacepattern provides for a substantially continuous network having voids andenhances the osteo-conductivity of the biomaterial and is a3-dimensional network. One example of the surface pattern can be derivedfrom FIG. 1.

More preferred is a method according to the present invention, whereinsaid embossing of the surface or surfaces of the biomaterial isperformed by rolling the material in a suitable roller press. Mostpreferred is a method according to the present invention, wherein voids(e.g. channels and indentations) are introduced having a medium widthranging from 30 microns as a minimal to widths approaching 230 micron incross dimension. As used herein, a void can be an opening through thesheet 2 or an indentation extending from the outer surface, but notthrough the sheet 2, the indentations can be hollow depressions or dipsor elongated channels all of which make up the network of voids.

In yet another aspect of the method according to the present inventionfurther said method comprises the subsequent and/or intermediate stepsof drying (e.g. freeze-drying) said biomaterial and/or storing of saidbiomaterial, preferably storing under vacuum (e.g. vacuum-packaged).

Yet another aspect of the present invention relates to a bone matrixscaffold comprising at least one sheet material as described above,wherein said at least one sheet material forms a macrostructure having asubstantially continuous network and voids as described above. In aparticular embodiment of the present invention, the inventive bonematrix scaffold comprises at least two sheets of material. Morepreferred is a bone matrix scaffold according to the present invention,comprising a laminate structure consisting of said at least two sheetsof material.

In another embodiment of the bone matrix scaffold according to thepresent invention, said network is semi-solid, in particular forming agel. Preferably, said bone matrix scaffold according to the presentinvention furthermore comprises crosslink-structures. More preferably,said crosslink-structures comprise at least one polymer and/or a mixtureof polymers. According to one embodiment of the bone matrix scaffoldaccording to the present invention said at least one polymer can becopolymer having carboxylic acid groups and/or amine groups. The atleast one polymer can be conductive polymer selected from polypyrrole,polyaniline, polyacetylene, and/or polythiophene. These conductivepolymers can be laminated in sheets with insulating layers between orinterpositioned with the conductive sheets to separate charge and definevoltage stacks in the poly-laminates. The insulating layers can bepolyurethane sheets or layers or a bioabsorbable insulation layer of anet neutral charge material.

Most preferred is a bone matrix scaffold according to the presentinvention, wherein said crosslink-structures comprise collagen fibers.Optimally, said crosslink-structures comprise non-aldehyde cross-linkedtype I collagen. In a particular embodiment of the bone matrix scaffoldaccording to the present invention, said crosslink-structures formspokes and/or mimic cell microfilaments. Schematic examples of thestructures of these particular embodiments can be taken from theaccompanying figures. In yet another particular embodiment of the bonematrix scaffold according to the present invention, wherein saidcollagen is chemically cross-linked with nordihydroguaiaretic acid orother suitable agents to confer stability.

The bone matrix scaffold according to the present invention can have asurface area of at least 1 m²/g. Preferably, the voids of saidmacrostructure of the bone matrix scaffold according to the presentinvention are substantially continuous. Thus, preferred is a bone matrixscaffold according to the present invention, wherein at least 10% ofsaid voids of said macrostructure are connected (are in contact with)said voids of said sheet material. Thus, a network is formed that can be3-dimensional throughout the bone matrix scaffold according to thepresent invention. Nevertheless, in other particular applications, itmight be suitable to provide a bone matrix scaffold according to thepresent invention, wherein said voids of said macrostructure and saidvoids of said sheet material are not connected.

In another embodiment of the bone matrix scaffold according to thepresent invention, said voids of said macrostructure define openings andthe average diameter of said openings and the average diameter of across-section of the network have a ratio of from 2:1 to 10:1. Preferredis a bone matrix scaffold according to the present invention, whereinthe ratio is from 2:1 to 5:1.

In yet another embodiment of the bone matrix scaffold according to thepresent invention, wherein the network of a cubic portion of said matrixhas dimensions of 0.5 cm on all sides and voids defining openings withan average diameter of 50-500 μm, and a connective number of at least10. Preferred is a bone matrix scaffold according to the presentinvention, wherein the connective number is at least 20.

In yet another embodiment of the bone matrix scaffold according to thepresent invention, wherein a cross section of said network has a maximumand minimum diameter, with a ratio of the maximum and minimum diameterof from 1:1 to 10:1.

Preferred is a bone matrix scaffold according to the present invention,wherein less than 10% of said voids of said macrostructure have afractal dimension higher than 1.

According to a preferred embodiment of the bone matrix scaffoldaccording to the present invention, the exterior surface of saidscaffold is porous. More preferably, the bone matrix scaffold accordingto the present invention has a porosity of at least about 20%.

Similar to the sheet material as above, also the bone matrix scaffold isessentially biodegradable, bioerodable and/or bioresorbable and/orbioinstructive. The same definitions as above apply.

According to a preferred embodiment of the bone matrix scaffoldaccording to the present invention, the bone matrix scaffold isessentially permeable to body fluids and/or cells. In yet another aspectof the bone matrix scaffold according to the present invention, the bonematrix scaffold further comprises cells and/or proteins. Said cells canbe selected from the group of living cells and recombinant cells,chondrocytes, growth factor producing cells, such as TGF FGF-producingcells, and osteoblasts, and the proteins are selected from proteins thatinhibit or enhance vascularization, enhance or retard innervation. Theseproteins are well known to the person of skill and described in theliterature, e.g. in Suzuki F. Roles of cartilage matrix proteins,chondromodulin-I and -II, in endochondral bone formation: a review.Connect Tissue Res. 1996; 35(1-4):303-7 and the references as citedtherein.

According to a preferred embodiment of the bone matrix scaffoldaccording to the present invention, the size of said matrix scaffoldchanges less than 50% when said cells are added to the matrix. Morepreferred, the bone matrix scaffold according to the present inventionhas a compressive modulus of at least 0.4 MPa at 4% strain, and/or adensity of less than about 0.150 g/cm³.

Another aspect of the present invention then relates to a method forproducing a bone matrix scaffold according to the present invention,comprising the steps of a) providing at least one sheet materialaccording to the present invention having a surface pattern thatenhances osteo-conductivity and potentially supports inductivity, thesurface pattern is a 3-dimensional substantially continuous networkhaving voids, and b) cross-linking said at least one sheet materialusing fibers, whereby a macrostructure is formed having a substantiallycontinuous network and voids. Said macrostructure or bone matrixscaffold that is formed is designated herein also as “cell” of the graftmaterial (different from the biological cells as also described herein)or “building block” of the graft of the present invention. In oneparticular embodiment, a building block comprises two sheets of materialthat are cross-linked by the method according to the present invention.In one particular embodiment of the method according to the presentinvention, a laminate structure consisting of said at least two sheetsof material is formed.

The method for producing a bone matrix scaffold according to the presentinvention in one embodiment forms a semi-solid network, in particular agel. Said gel can consist of either a laminate structure or multiplebuilding blocks as described above. More preferred is a method forproducing a bone matrix scaffold according to the present invention,wherein said fibers comprise at least one polymer and/or a mixture ofpolymers. In one embodiment of the method for producing a bone matrixscaffold according to the present invention, said at least one polymeris a copolymer having carboxylic acid groups and/or amine groups. Mostpreferred is a conductive polymer selected from polypyrrole,polyaniline, polyacetylene, and polythiophene and mixtures thereof.These conductive polymers can be laminated in sheets with insulatinglayers between or interpositioned with the conductive sheets to separatecharge and define voltage stacks in the poly-laminates. The insulatinglayers can be polyurethane sheets or layers or a bioabsorbableinsulation layer of a net neutral charge material.

Preferred is a method for producing a bone matrix scaffold according tothe present invention, wherein said fibers are collagen fibers, inparticular non-aldehyde cross-linked type I collagen. According to amore preferred method for producing a bone matrix scaffold according tothe present invention, said collagen is chemically cross-linked, inparticular with nordihydroguaiaretic acid or other suitable agents toconfer stability.

In yet another embodiment of the method for producing a bone matrixscaffold according to the present invention, said voids are formed todefine openings and the average diameter of said openings and theaverage diameter of a cross-section of said network is formed to have aratio of from 2:1 to 10:1, preferably said ratio is from 2:1 to 5:1.

In yet another embodiment of the method for producing a bone matrixscaffold according to the present invention, less than 10% of said voidsof said macrostructure are formed to have a fractal dimension higherthan 1.

Preferred is furthermore a method for producing a bone matrix scaffoldaccording to the present invention, wherein the exterior surface of saidscaffold is made porous.

Yet another aspect of the method for producing a bone matrix scaffoldaccording to the present invention further comprises the step ofincubating said macrostructure with cells for a predetermined period oftime. To assure that the matrix deposited on the scaffold is bonespecific, both osteoblasts and mesenchymal stem cells preferably areused to further create the bio-scaffold matrix. In choosing a cell linesuitable for providing the osteoinductivity of the deposited matrix, itis important that the cells do not in normal culturing conditionsspontaneously differentiate into osteoblasts or themselves produce anybone-specific proteins (e.g. alkaline phosphatase). While it is notsurprisingly that DBM induces differentiation of confluent humanperiosteal cells into osteoblast-like cells, the true test ofinductivity is demonstrated in that DBM exposure also producesbone-specific response in skeletal muscle myoblast culture (see alsobelow).

Preferred is furthermore a method for producing a bone matrix scaffoldaccording to the present invention, wherein said cells can be selectedfrom the group of living cells and recombinant cells, chondrocytes,growth factor producing cells, such as TGF FGF-producing cells, andosteoblasts, and the proteins are selected from proteins that inhibit orenhance vascularization, enhance or retard innervation. These proteinsare well known to the person of skill and described in the literature,e.g. in Suzuki F. Roles of cartilage matrix proteins, chondromodulin-Iand -II, in endochondral bone formation: a review. Connect Tissue Res.1996; 35(1-4):303-7 and the references as cited therein. In stillanother embodiment, the distillate of material lypholization iscollected, and microprinted back on the organic matrix with specificattention to the shape, dimensions, voids, so that a biological imprintof specific design is used to define cell attachment and guide tissueregeneration

In another preferred method for producing a bone matrix scaffoldaccording to the present invention, the size of said macrostructurechanges less than 50% when said cells are added to the matrix.

Yet another preferred method for producing a bone matrix scaffoldaccording to the present invention further comprises removing said cellsfrom said macrostructure, in order to produce a bone matrix scaffoldthat can be either used directly, forms an intermediate for otherproducts, is shaped by a suitable technique (as described below for thegraft), and/or is used in order to produce a final bone graft, asdescribed below.

Another aspect of the present invention then relates to a bone graft,comprising at least one bone matrix scaffold according to the presentinvention and osseous and/or chondrial material. In one embodiment, saidbone graft according to the present invention comprises at least twobone matrix scaffolds being connected by fibers. Preferably, said fiberscomprise at least one polymer and/or a mixture of polymers, morepreferably the at least one polymer is a copolymer having carboxylicacid groups and/or amine groups. Most preferred is a bone graftaccording to the present invention, wherein said at least one polymer isa conductive polymer selected from polypyrrole, polyaniline,polyacetylene, and polythiophene and mixtures thereof.

In yet another embodiment of the bone graft according to the presentinvention, said fibers are collagen fibers, preferably said fibers arenon-aldehyde cross-linked type I collagen. If the fibers arecross-linked, the bone graft according to the present inventioncomprises collagen that is chemically cross-linked, in particular withnordihydroguaiaretic acid (NDGA).

According to yet another embodiment of the bone graft according to thepresent invention, said graft is present in the form of a facial andcosmetic surgery graft. Furthermore, the bone graft according to thepresent invention can be an allograft or autograft.

A preferred bone graft according to the present invention consistsessentially of osseous material and/or chondral material. Nevertheless,a bone graft according to the present invention can further compriseosteoinductive substances, preferably, said osteoinductive substancesare bone specific growth factors as described herein and known to theperson of skill, e.g. from Gamradt S C, Lieberman J R. Geneticmodification of stem cells to enhance bone repair. Ann Biomed Eng. 2004January; 32(1):136-47, and Sammarco V J, Chang L. Modern issues in bonegraft substitutes and advances in bone tissue technology. Foot AnkleClin. 2002 March; 7(1):19-41 and the references as cited therein.

Yet another aspect of the present invention relates to a method forproducing a bone graft according to the present invention, comprisingthe steps of a) providing at least two bone matrix scaffolds accordingto the present invention, b) connecting (e.g. cross-linking) said atleast two bone matrix scaffolds using fibers, and c) depositing ofosseous material and/or chondral material on said connected at least twobone matrix scaffolds.

Preferred is a method for producing a bone graft according to thepresent invention, wherein said fibers comprise at least one polymerand/or a mixture of polymers, particularly at least one polymer is acopolymer having carboxylic acid groups and/or amine groups, morepreferably said at least one polymer is a conductive polymer selectedfrom polypyrrole, polyaniline, polyacetylene, and polythiophene andmixtures thereof.

In yet another aspect of the method for producing a bone graft accordingto the present invention, said fibers are collagen fibers, preferablysaid fibers non-aldehyde cross-linked type I collagen. In a preferredmethod for producing a bone graft according to the present invention,said collagen is chemically cross-linked, in particular withnordihydroguaiaretic acid.

In yet another aspect of the method for producing a bone graft accordingto the present invention, said depositing of osseous and/or chondrialmaterial comprises incubating said connected at least two bone matrixscaffolds with cells for a predetermined period of time. Said cells canbe selected from the group of living cells and recombinant cells,chondrocytes, growth factor producing cells, such as TGF orFGF-producing cells, and osteoblasts, and the proteins are selected fromproteins that inhibit or enhance vascularization, enhance or retardinnervation. These proteins are well known to the person of skill anddescribed in the literature, e.g. in Suzuki F. Roles of cartilage matrixproteins, chondromodulin-I and -II, in endochondral bone formation: areview. Connect Tissue Res. 1996; 35(1-4):303-7 and the references ascited therein.

A preferred method for producing a bone graft according to the presentinvention furthermore comprises removing said cells in order to producea bone graft that can be either used directly, forms an intermediate forother bone-related products, and/or is shaped by a suitable technique(as described below).

Yet another preferred method for producing a bone graft according to thepresent invention, further comprises shaping the graft, preferably by amethod comprising CAD, cutting, moulding and/or pressing. Morepreferably, the graft is shaped in the form of a facial and/or cosmeticsurgery graft. Most preferred is a method for producing a bone graftaccording to the present invention, wherein the graft is shaped in apatient specific form.

Another method for producing a bone graft according to the presentinvention further comprises providing osteoinductive substances, inparticular proteins, to said graft. Examples are indicated above.Preferably, said osteoinductive substances are bone specific growthfactors.

Another aspect of the present invention is then related to a method forsurgical, plastic and/or cosmetic bone replacement for a patient,comprising a) providing a graft according to the present invention, andb) implanting said graft into said patient. In said method according tothe present invention, the implanting can be performed during cartilage,bone, joint, and/or articular cartilage repair and/or replacement. In apreferred embodiment of the method according to the present invention,said implanting comprises the direct implantation into the matrix of thebone to be replaced.

Preferred is a method for surgical, plastic and/or cosmetic bonereplacement according to the present invention, wherein said providingof said graft further comprises shaping the graft, preferably by amethod comprising CAD, cutting, moulding and/or pressing. Morepreferably, the graft is shaped in the form of a facial and/or cosmeticsurgery graft. Most preferred is a method for surgical, plastic and/orcosmetic bone replacement according to the present invention, whereinthe graft is shaped in a patient specific form, which can then readilybe used for the bone replacement treatment. Alternatively, one can usethe material as a drug delivery, or cell differentiation incubator. Theability for the poly-laminates to hold specific charge can create alatent charge that functions as a differentiation device, in process,cylinders of porous material, interlaid with autologous patient cellswould be implanted in bone, in muscle flaps, in areas of vascular flowand the inherent material charge would not only summon stem cells butpartition their pathway towards specific phenotypic expression.

A last aspect of the present invention is then related to a device forproducing a sheet material according to the present invention,comprising a device that embosses, etches and/or cuts anosteo-conductive 3-dimensional surface pattern having substantiallycontinuous network having voids into a suitable sheet material. In apreferred embodiment of the present invention, the device according tothe present invention comprises compressing rollers.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described by way of example and with reference to theaccompanying drawings in which:

FIG. 1 shows a type-I collagen matrix cut by free-electron laser toreproduce cancellous bone according to the state of the art.

FIG. 2 shows a collagen scaffold representing cancellous bone accordingto the invention, wherein a tile pattern has been used.

FIG. 2A is a photo reproduction of cancellous whale bone, the largeportion being of the same scale as shown in FIG. 2, the upper left viewbeing at scale, the lower left view being magnified.

FIGS. 3A and 3B show two longitudinal sections of allograft fibermaterial after 3 and 6 weeks of culturing with cells according to thestate of the art. Differing in magnification, no apparent difference insize was evident despite the much thicker matrix that attached to theNDGA-treated fibrils.

FIG. 4 shows a chart of the fibroblast attachment to tissue-culturetreated dishes coated with collagen, and collagen-coated dishes treatedwith NDGA. The number of cells attached following removal of unattachedcells with Dulbecco's PBS measured with CyQuant cell proliferationassay.

FIG. 5 is a schematic representation of a laminate structured sheetmaterial having different (controlled) degradability.

FIG. 6 is a schematic representation of one preferred embodiment of theinvention. Two sheet materials are connected by fibers.

FIG. 7 is a schematic representation of another preferred embodiment ofthe invention, wherein the void between the sheet material is controlledby the fibers.

FIG. 8 depicts a schematic representation of another preferredembodiment of the invention, wherein a stack of bone matrix scaffoldsaccording to the present invention is shown.

FIG. 9 schematically shows a method for directly implanting the deviceaccording to the present invention.

FIG. 10A-10D are schematic drawings of yet another embodiment showinglaminates being shaped and connected by interposed tissue blocks.

FIG. 11 shows the schematic drawing of an inventive tube-shapedembodiment for bonding and binding two loose ends of tendon material.

FIG. 12 shows a schematic drawing of a device for producing a sheetmaterial according to the present invention by rolling.

FIG. 13 shows another schematic drawing of the device according to FIG.12, further equipped for producing thin wafer material for producing asheet material laminate.

DETAILED DESCRIPTION OF THE INVENTION

Free-electron Laser etching was used to attempt replication ofcancellous bone on varying substrates applied to cell culture plates.The underlying hypothesis, analogous to “building a brick wall”,proposes that after setting the first tiers, optimal structure resultsfrom subsequent bricks following a precedent of pattern. In thisparadigm, the trabecular scaffold represents the foundation of pattern.Although the material 100 was encouraging, insufficient resolution ofthe laser prevented reproducible fabrication, an important considerationin developing cancellous bone as a manufactured allograft where thestructure is intended to confer both physiological and physicalcharacteristics, as illustrated in FIG. 1.

The surface pattern is introduced by etching and/or embossing thesurface or surfaces of the biomaterial. The surface pattern provides fora substantially continuous network having voids and enhances theosteo-conductivity of the biomaterial and is a 3-dimensional network.One example of the surface pattern can be derived from FIG. 1. Forcomparison, FIG. 2A shows a photo of an actual whale bone 100W of thesame scale as FIG. 2. The voids and ridges of the actual whale bone 100Wremarkably similar to the replication pattern 100A of FIG. 2. The upperleft small photo is to scale; the lower left is an enlargement. Thisability to mimic a natural and successful bone pattern is believed to beextremely beneficial to achieving enhanced new bone growth on boneimplant devices made of plastic or metal implantable materials.

Advancements in technology and refinements in application now permitreproducible templates 100A of collagen to be made with 10-micronresolution. Based on high resolution micro-CT analysis of blocks ofwhale bone (Microphotonics, Inc., Allentown, Pa.), and Micro-CT Center,UCONN Health Center), planar stacks of material 10 can now bereproducibly made that replicate the cancellous morphology ofunder-modelled mammalian bone, as shown in FIG. 2, using template 100Adriven compression molding derived from patterns 100A detailed(Intelligent Micropatterning, LLC, St. Petersburg, Fla.) and etchingsdefined in metal masters rollers 44, 46 (Akron Metal Etching, Co.,Akron, Ohio) as shown in FIGS. 12 and 13 material 40 can be achievedrepeatedly in sheets or layers 40.

This application in one embodiment utilizes type-I collagen matricesthat have been cross-linked by a unique process to provide a suitablescaffold for culturing of osteoblasts. The uses of structured allograft,or of the structural properties of the allograft material are considereda primary basis for this technology. As a structural property, theshape, confluence, connectivity, density, porosity and cell stimulationcharacteristics are considered valuable. Bone is a structure where formdictates functional efficiency. Knowing that bone forms underdistraction (tension), it also models under compression. The openarchitecture of this novel biomaterial is to produce bone specificmatrix during incubation, and effect efficient tension at the cellmatrix interface to support osteoblast physiology.

EXAMPLE 1 Collagen Preparation

Human placentas that have been screened for HIV and Hepatitis preferablyare used as the source of collagen. The preferred human fetal membranespreferably are insoluble amnion, soluble amnion, soluble chorion orcombinations of them. Both the amnion and the chorion preferably arecleaned of any blood clots or debris. Collagen preferably is extractedusing limited proteolytic digestion with pepsin. In brief, tissuepreferably is homogenized in 0.5 M acetic acid, the pH adjusted to 2.5with HCl and the preparation digested twice with pepsin (10 mg pepsin/gmwet weight tissue) overnight. A combination method of selectiveprecipitation from neutral salt solvent and acid solvents preferably areused to purify the collagen. Purified collagen preferably isreconstituted by dialysis against low ionic strength sodium phosphatebuffer (pH 7.2) at 15-17° C. The purity of the type-I collagenpreferably is assessed by SDS/PAGE using 4-20% linear gradientTris-glycine gels (Koob T. J. and Hernandez D. J., Biomaterials; 23:203-212, 2002).

Collagen preferably is produced under semi-confined conditions to yielda rectangular sheet of material 40 approximating 1×2 cm×150 micron inthickness. This sheet 40 of biomaterial will then be run through aroller press 44, 46 that will simultaneously emboss the upper and lowersurfaces of the collagen with the prescribed porosity and geometry ofunder modelled bone 100A as shown in FIG. 12. This material preferablyis further dried and stored under vacuum.

NDGA Cross-linking: Modelled collagen sheets preferably are hydrated in01.M NaH₂PO₄, 0.15M NaCl, pH 7.0 (PBS) for 30 minutes followed bytreatment with 30 mg/ml NDGA (Sigma Chemical Co., St. Louis, Mo.) asfollows: NDGA preferably is suspended in 1N NaOH; and then added to thePBS to achieve a final concentration of 3 mg/ml. The collagen sheetspreferably are agitated in solution for 24 hours at room temperature,washed in 70% ethanol in water followed by extensive washing with PBSand the NDGA process preferably are repeated. NDGA appears to have noaffect on cell attachment with regard to cultured fibroblasts oncollagen vs. NDGA-treated collagen as shown in FIG. 4. Directmeasurement in osteoblast cultures has not been performed.

In other studies to assess the ability for cells to migrate andproliferate in explant conditions, NDGA-cross-linked fibers wereextended through extensor tendons and at specific incubation times up to9 weeks and assayed for cell number. The number of cells increasedthroughout the incubation period independent of the cross-linkingtechnology, and the closer to the source of the tissue the greater thenumber of cells that could be measured (Koob T. J. and Willis T. A., J.Biomed. Mat. Res.; 56: 40-48, 2001).

Excipient Technology: Bioactive factors are critical to influencing thedirection, sequence and speed of the regenerative process. Extensiveanimal and strong clinical data support that osteoinductive growthfactors in appropriate dose are sufficient in themselves to effect abiologic response and stimulate bone regeneration. A recent FDA approvalof the InFUSE bone graft device would be case in point for data,approval, and regulation of recombinant factors as a device (Departmentof Health and Human Services, 2. July 2002;http://www.fda.gov/cdrh/mda/does/p000058.pdf).

The inventive scaffold 2 mirrors the surface properties of cancellousbone 100A as a synthetic analogue fashioned from cross-linked humancollagen to enhance cell attachment and matrix deposition, and attenuatematrix factors in deposit that are in themselves osteoinductive throughosteoblast priming Matrix priming, or “pharming”, can be extended inapplication as both a repository and a delivery device, utilizingcell-based delivery systems to effect composition without confoundingthe delivery with a cell-based system. Through genetic engineering, itis possible to obtain bone matrix tailored to intent without theencumbrance of cells in the delivery system. This strategy profoundlyaffects the regulatory pathway for gaining market approval andexpediting patient treatment. The application teaches to manufacture amineralized bone matrix that could be used in place of current DMBproducts, while other devices embody multi-planar laminates 10 ofcultured bone sheets 40 to confer 3-D structure. These structurespreferably are modular and can be fitted for filing osseous defects inreconstructive efforts, or used as biologic bridges for restoringcontinuity in defects.

To assure that the matrix deposited on the scaffold 2 and/or sheetmaterial 40 is bone specific, both osteoblasts and mesenchymal stemcells preferably are used to create the bioscaffold 2 matrix. Inchoosing a cell line suitable for assaying the osteoinductivity of thedeposited matrix, it is important that the cells do not in normalculturing conditions spontaneously differentiate into osteoblasts orthemselves produce any bone-specific proteins (e.g. alkalinephosphatase). While it is not surprisingly that DBM inducesdifferentiation of confluent human periosteal cells into osteoblast-likecells, the true test of inductivity is demonstrated in that DBM exposurealso produces bone-specific response in skeletal muscle myoblastculture.

To validate that the manufactured matrices 2 are engendered with acapacity for osteoinductivity, skeletal muscle myoblast cell linespreferably are used to measure differentiation towards an osteoblastphenotype on the decalcified matrix. The presence of bone-specificproteins during subsequent incubation preferably are interpreted as aresponse to matrix factors present in the manufactured bone scaffold.Previous work has demonstrated the advantage of spinner flask cultureover static culture at 14 and 21 days, in particular enhancedmineralization by convection culturing (Sikavitsas V. I., et al., J.Biomed. Mat. Res.; 62: 136-146, 2002).

Cell culture: Human Osteoblast Cells preferably are obtained from Lonza(Walkersville, Md.) and cultured in alpha-MEM (Gibco/BRL #12561-023)with 1% Pen/Strep (Gibco/BRL=15140-015) and 10% FBS (Hyclone #A-1115-L)at 37° C. in 5% CO₂. Cells preferably are subcultured every 3-4 days asfollows. Cells preferably are washed twice with 5 ml Hanks balanced saltsolution without Ca⁻ or Mg⁻ (BioWhittaker #10-547F, now a division ofLonza) that has been pre-warmed to 37° C. Hanks solution preferably isaspirated and then 2 ml of 0.001% pronase incubated with the cells for 5minutes at 37° C. Volume preferably is brought to 10 ml with pre-warmedalpha-MEM and a pipette used to dissociate the cells. Cells will then besplit 1:10 and carried for additional growth. Induction of phenotype(mineralization) preferably is accomplished by supplementing the mediawith Hydrocortisone 21 Hemisuccinate and β-glycerophosphate as suggestedby cell line supplier.

Mesenchymal stem cells (Human Bone Marrow) will also be obtained fromLonza that have been tested for purity by flow cytometry and for theirability to differentiate into osteogenic, chondrogenic and adipogeniclineages. Cells are positive for DC105, CD166, CD29, and CD44. Cellstest negative for CD14, CD34 and CD45. Media systems specific to supportgrowth of hMSCs and their differentiation into osteogenic lineagepreferably is obtained from the supplier.

Normal human muscle myoblasts (HSMM) preferably is similarly obtainedfrom Clonetics. The cell and media support system advocated by thesupplier preferably are used to generate HSMM cultures for the study ofcellular development and differentiation. By using three distinct celllines, it preferably is possible to perform a detailed analysis ofsynergistic influence of prepared scaffold on mesenchymal cells, andalso allow a pure osteoinductivity experiment on non-skeletal cell linesto assay for differentiation and induction.

Cultures preferably are enzymatically lifted from T-75 flasks with 4 mlof trypsin-EDTA solution (0.05% trypsin; Gibco BRL) when they reach70-80% confluency. Cells preferably are counted and added in a cellsuspension drop-wise to the polymer scaffolds. The volume of the cellsuspension added to each scaffold preferably is approximately 1 ml,having a cell density of 10⁶ cells/ml. Cells preferably are allowed toadhere to the cell-scaffold constructs for 2 hours and then pieced into6-well plates and covered with 10 ml of media overnight in the incubatorbefore being placed in the bioreactor. Osteogenic media preferably issupplemented with 100 nM dexamethasone; 10 mM β-glycerophosphate; and 50μg/ml ascorbic acid-2-phophate (all from Sigma) (Jaiswal N., et al., J.Cell Biochem.; 64: 295-312, 1997).

The spinner flask system consists preferably of a dual side armcylindrical flask with a diameter of 58 mm (Belco, Vineland, N.J.) and aNo. 12 rubber stopper which will serve as a cover. Three pairs ofcell/scaffold constructs preferably are suspended centrally within thespinner flasks. A volume of approximately 120 ml of osteogenic mediapreferably is added to completely cover the scaffolds, and magneticstirrer at the bottom of the flask will maintain 30 rpm. Media in allcultures preferably is changed every 2-3 days. Cultures preferably areevaluated at 7, 14, and 21 days for DNA content, Alkaline Phosphatase(ALP) activity, osteocalcin secretion, for calcium deposition and byhistology for bone matrix formation.

Analysis: The study can preferably have 4 arms and evaluate the culturedscaffold/cell constructs at time intervals of 7, 14, and 21 days,evaluating: human osteoblasts with fabricated scaffold 2; humanmesenchymal stem cells on fabricated scaffold 2; human mesenchymal stemcells on retrieved scaffold 2; human skeletal muscle cells on retrievedscaffold 2 from osteoblast culture.

DNA Measurement: Cellularity of the cell/scaffold constructs preferablyis determined using a fluorometric DNA assay (West D. C., et al., Anal.Biochem.; 147: 289-295, 1985). Briefly, scaffolds preferably are removedfrom the bioreactors at day 7, 14, or 21, washed with double distilledH₂O, and homogenized in 1.4 ml of cold 10 mM EDTA, pH 12.3. Thehomogenates preferably are sonicated for 10 minutes in an ice bath,incubated for 20 minutes at 37° and returned to an ice bath. A volume of200 μl of 1 M KH₂PO₄ preferably is added to neutralize the pH. DNAstandards preferably are prepared from stock DNA solutions containinghighly polymerized calf thymus DNA (type I, Sigma) at a concentration of50 μg/ml. A volume of 200 μl of the standard or the homogenized samplepreferably is mixed with 1.3 ml of a 200 ng/ml Hoechst 33258-dye(Polysciences, Warrington, Pa.) in a 100 mM NaCl and 10 mM Tris buffersolution. The fluorescence emission at 455 nm preferably is read at anexcitation wavelength of 350 nm on a fluorescence spectrophotometer.

ALP activity: AP activity preferably is measured with a commerciallyavailable kit (ALP-10, Sigma). Scaffolds preferably are placed incentrifuge tube containing 1 ml of a 1M Tris solution at neutral pH andhomogenized. The homogenate preferably is added to 1 ml of reconstitutedreagent provided by the kit at 30° C. Absorbance preferably is measuredevery minute for 4 minutes at 405 nm using a HP 8452A Diode arrayspectrophotometer. The slope of the absorbance versus time preferably isused to calculate the ALP activity.

Osteocalcin secretion: Osteocalcin secreted in the culture mediapreferably is determined using a commercially available sandwichimmunoassay (BT-480) from BTI (Stoughton, Mass.). The BTI Mid-TactOsteocalcin Elisa Kit is highly specific. It measures both the intacthuman osteocalcin and the major (1-43) fragment. The assay is a sandwichELISA which employs two monoclonal antibodies. One antibody (1-19) isimmobilized in the wells and the second antibody (30-40) isbiotinylated. The assay is highly sensitive (0.5 ng/ml) and requiresonly a 25 microliter sample. All the necessary reagents, a 96-well stripplate, and a complete 3½ hour protocol are included with the kit.

Calcium deposition: Calcium deposition on the scaffolds preferably aremeasured by the ortho-cresolphtalein complexone procedure (SigmaDiagnostics, Procedure No. 587). Scaffolds preferably are washed withdistilled water, and placed on an orbital shaker to incubate overnightin the presence of 2 ml of 0.5 N acetic acid. Equal volumes of thecalcium-binding reagent (0.024% orthocresophtalein complexone and 0.25%8-hydroxyquinalone) and the calcium buffer (500 mmol/l2-amino-2-methyl-1,3 propanediol and other non-reactive stabilizers)provided in the assay kit preferably are mixed to generate the assayworking solution. A volume of 300 μL of working solution preferably isadded to 10 μl of sample in a 96-well plate. To generate a standardcurve, serial dilutions of CaCl₂ preferably are prepared (1-250 μg/ml).The plate preferably is incubated at room temperature for 10 minutes andthen read at 575 nm. Calcium deposition from each scaffold preferably isreported as mg Ca²⁺ equivalents.

Histology and tetracycline fluorescence microscopy: Scaffolds 2preferably are immersion fixed in 2% glutaraldehyde, dehydrated inrising concentrations of alcohol and rapidly embedded into plastic forthin sectioning. Sections preferably are stained by Goldner trichromeand Toluidine blue methodology. Mineral deposition preferably isevaluated by adding tetracycline-HCL in the culture media at a finalconcentration of 10 μg/ml and is a well-established methodology forevaluating matrix deposition. Tetracycline accumulates at bone formingsites and morphometric evaluation preferably is carried out usingstandard Bioquant software on a Nikon E1000 research microscope.

This example demonstrates the utility of imbuing precision scaffoldswith bone specific matrices that could be substituted for humanallograft. Conventional strategy for developing repair material for bonehas long sought to duplicate the biomechanical characteristics of bonein order to enhance a seamless integration and achieve a rapidrestitution of function. In addition to the use of allograft andautograft, substitute matrices of coral, bovine bone and a variety ofpolymers have been evaluated. Only recently have efforts shifted toseeding osteoblast facilitate attachment. Various shortcomings haveaccompanied each effort, ranging from poor absorption and fear of viraldisease, through outright tissue incompatibility. Regulatory issues forcell-mediated therapies and growth factor delivery also have slowedclinical applications.

The invention shows a departure from those efforts, in that it seeks tospecify a scaffold 2 that has been exposed over its course ofdevelopments to microgravity. As such, bone cell attachment, loading,and integration will all respond to the dynamic tension of subsequentcompressive loading because of it engineered structure, while at thesame time the inherent porosity of the scaffold will facilitate matrixenhancement. Fabricating a scaffold carries with it an intrinsicopportunity for biomolecular engineering to manipulate tissue phenotype,or to combine therapeutic additions to normal bone matrix. Using a humancollagen matrix that has been cross-linked to assure static structure inculture will enhance the material properties that preferably arestimulated by loading.

EXAMPLE 2

FIG. 5 shows a poly-laminate of material 10 with specified shape and ofpre-determined degradability. In a series of laminar structures withincreasing resistance to degradation and with continuity of integraldesign, a plating system that allowed for not only bridging but also forexpansion is envisioned. Given known material thickness and the abilityto reconstruct from normal materials using high resolution CT, it is nowpossible to formulate materials that have specific construction and inpurpose simulate under structured bone 100A.

EXAMPLE 3

The scaffold 2 can be used as a primer for bone formation. Currentcadaver sources of allograft bone are insufficient, and revolve aroundissues of contamination and the mores of harvesting donor tissue. Whilein principal, profiting from cadaver material is not allowed,not-for-profit operations may have significant operating costs thataffect the price-to-patient for use. Alternative sources of autograftare limited in quantity and require operative treatment to obtain. Inthis invention bio-conductive monolayer matrices 2 preferably arecultured under optimized conditions that result in bone production andmineralization. Materials are then bonded by effective polymerization tocreate solid materials with known porosity and predictable properties ofbone conductivity and inductivity. Cells preferably are removed from theprocess and the bulk material treated to remove allogeneic components asif it were a “donor” material 2. As a material without cells, the FDAcan treat this material as a device, and it should be available for a510K regulatory status. Cells can be endowed with specific geneticcomponents that will result in enhanced deposition of specific proteins.Proteins such as those in the TGF super family, basic transductionsequences such as the Lim promoter, and other families of growth factorswould be considered. As the specific protein of the growth factor ratherthan the gene for production would be lending itself to the composition,the value of a matrix that had been enhanced would be the consideration.Specific proteins that inhibit vascularization, enhance vascularization,enhance innervation, retard innervation, and other substances that couldbe secreted are considered.

EXAMPLE 4

For use in articular cartilage repair, the use of structured matrix 2with defined porosity is considered, as illustrated in FIGS. 5 and 6.Chondrocytes of known genetic make-up preferably are used to define thematrix 2 that is produced. Cells preferably are removed, and matrixpreferably is available as generated gel substance.

Each plate 10 would be connected by a series of collagenous fibers 12that produce tension like spokes on a bicycle or mimics cellmicrofilaments for supporting cell wall. The FIGS. 5 and 6 represent one“cell” in the construct. Between the parallel plates 10, cells would beallowed to produce matrix 2, and then by cutting fixed templates(trephine, punch-type) defined matrix 2 components could be engineeredto size. Heights would be adjustable by the length of the fiber systems12 used to construct. By seeding with matrix 2, or by creating materialsfor articulation, this scaffold would permit programmable obsolescence,enhancing progressive load sharing.

The fixture of claim is the use of micro-engineered laminates 2 ofspecified materials that use a cell-based protein production of matrix,as illustrated in FIG. 8. Using both gene-enhanced systems, and inspecific conformation, the goal is to engineer a more conductive, moreintegrative, stronger and more efficient biologic system. Such design isnot limited to either cartilage or to bone systems and the value increating other specific organs is intended as well. The basic polymer,or collagenous materials could be porous, have sieving propertieslimited to molecular size and would have defined flexibility. At thecurrent time, degradability is considered an asset in progressive loadsharing.

EXAMPLE 5

As a fixture for containing autologous cells, the above poly-laminate 2is considered with defined porous volume, known biomechanical capacity,and an inherent ability to contain cells as shown in FIG. 9. One of thekey issues of autologous cell transplantation has been the lack ofappropriate containment device. Periosteal covers require additionalsurgeries, and strategies to directly inject multi-potent stem cellsinto joints have met with mixed support. In a preferred embodiment ofthe method according to the present invention, said implanting comprisesthe direct implantation into the matrix of the bone to be replaced asshown in FIG. 9.

EXAMPLE 6

The ability to press the laminate 2 and to define form in size and shapeis a separate consideration. In defining a series of sheets 10 or 40 ofbone, is then possible to press the sheets together to gain a definedform. Defined constructs for facial and cosmetic surgery come readily tomind, and the lack of compatibility issues, the strong role of inductivesubstances in structure of know micromorphometry, and the off-the-shelfcapacity for the substance to integrate make this an ancillary andvaluable option. Block materials could either shaped in surgery todefined CAD specifications, or pre-pressed shapes could be utilized asschematically illustrated in FIGS. 10A-10D. In consideration formaterial constructs, interlocking blocks 22, 23, 24 of tissues could beassembled (for instance like Lego® block Systems) to create appropriateand approximate contours, using rounded cap 25 pieces to fill the finaldimensions of staggered objects. In this consideration, caps 24 andshoulder 26 pieces would be fashioned as well. While the examples inFIGS. 10A-10D are essentially geometric, the shoulders 26 inmanufacturing would be best considered arcs to take advantage of thesmooth contours. Limitations of the sketches are those of the artist.

EXAMPLE 7

A use of material 2 that takes advantage of the properties implies inthe tensile tube. Similar to the “Chinese Handcuffs” this would allowthe fashioning of collagen fibrils 2 in the braided manner to create amethod of bonding and binding two loose ends of tendon material 14, 16as shown in FIG. 11. The material would be deployed as a loose mesh offixed diameter in flaccid state, and when pulled would cinch about thefree ends of the tendon to effect a manner of binding. Essentially thisis a flat braid that is self-tightening. It could be fashioned with acentral hollow pleating structure to offer purchase of the two free endsand to allow the outer shroud of material 2 to be deployed.

EXAMPLE 8

Devices for producing laminate microstructures according to theinvention are illustrated in FIGS. 12 and 13. A last aspect of thepresent invention is then related to a device for producing a sheetmaterial according to the present invention, comprising a device thatembosses, etches and/or cuts an osteo-conductive 3-dimensional surfacepattern having substantially continuous network having voids into asuitable sheet material. In a preferred embodiment of the presentinvention, the device according to the present invention comprisescompressing rollers as shown in FIGS. 12 and 13. The followingdescription is designed to impart a low-cost efficient use of patterningfor making laminate microconstructs. A trademark of Osteotech, Grafton®,a commercially available morselized grafting material or other suitableprocessed allograft materials are run through a roller 44, 46, much thesame as a wringer-type washer apparatus. The rollers 44, 46 are ofsufficient hardness as to create and indented surface 100A similar to apasta cutter, in essence a particular porosity in laminate 40 is achieveby offsetting die-cut mechanisms of the surfaces. This extruding devicecould be constructed of hard plastic or often suitable material(disposable, sterilizable) and be for immediate use in the operativetheatre. It would be lubricated by patient serum and would use the serumadherent as a self adhesive. By using non-proprietary products onapproved products, it preferably is possible to more quickly come tomarket with a designed material. Each roller 44, 46 exists as anembossing press that engenders a pattern 100A of cancellous bone inaddition to reducing the sheet 40 to a thin wafer that preferably ispart of a laminate 40. Poly-laminate of embossed osteo-inductivematerial 2. The invention might consist of one or more in a series ofrollers 44, 46 that will reduce to a thin size of appropriate porosity,followed by a second set of rollers 44, 46 that will cut in proportionto the diameter of the wheel so that a series of laminates 40 of knownarchitecture can be stacked. Separate from the matrix components 2 thisis considered a mechanical device. It would be considered a positiveendowment to have serum pushed into the matrix during the initialpressing and them for the porosity developed in the stacking to create amore inductive material 2.

Table 2 is the morphometric data of human cancellous bone samples H-1-H4and whale cancellous bone W1. Briefly the entire specimen was imaged andthe whale bone was purposely cut large to look for the internalconsistency of the form to follow variation in scales of sizing. Thecancellous bone samples range from 1-4 also in order of being mostosteoporotic (1) and the number (4) specimen being the most normal bone.Number 3 specimen is likely an outlier and might sit adjacent to acortical margin. The whale bone is consistent independent of boundaryrange or isometric randomization to size. The value in the whale bone isto isotropic distribution, thicker trabecula, greater trabecularspacing, and highest tissue density with lowest connectivity forequalized total volume. The importance is ridge dynamics, higher densitywith lesser void despite having greater separation makes this an idealpattern for mimicking to enhance new bone growth in humans.

TABLE 2 BVF Trab. Trab. Trab. Apparent Tissue Total Bone Bone Sam- (BV/Thick- Num- Spac- Connec. Density Density Vol- Vol- Sur- ple TV) nessber ing Density mg/ccm mg/ccm ume ume face BS/BV BS/TV BS/MV No. % μm1/mm μm 1/mm{circumflex over ( )}3 HA HA mm{circumflex over ( )}3mm{circumflex over ( )}3 SMI mm{circumflex over ( )}2 mm{circumflex over( )}2 1/mm 1/mm DA Just within boundaries of pieces H1 14.6% 142 1.41653 6 65 888 570.999 83.594 1.7 1595.7 19.0 2.8 3.3 1.6 H2 21.0% 1461.69 521 7 149 869 541.047 113.830 0.8 1989.8 17.2 3.7 4.7 2.8 H3 24.0%191 1.65 525 6 185 917 417.510 100.217 1.0 1399.6 13.8 3.4 4.4 1.6 H431.7% 156 2.24 374 17 286 891 380.271 120.339 0.3 1908.7 15.6 5.0 7.31.7 W1 21.1% 183 1.20 800 3 190 866 1070.286 225.320 0.4 3126.6 13.7 2.93.7 1.4 Smaller isometric cube ROI H1 15.9% 139 1.63 578 6 87 890 70.11311.152 1.9 208.8 18.9 3.0 3.5 1.6 H2 24.0% 150 1.80 488 8 186 871 70.11316.798 0.8 277.5 16.4 4.0 5.2 2.4 H3 26.3% 174 1.78 487 8 218 911 70.11318.451 0.9 266.0 14.4 3.8 5.1 1.7 H4 34.6% 156 2.36 360 18 319 88270.113 24.256 0.0 372.6 15.2 5.3 8.1 1.7 W1 21.1% 167 1.29 768 3 226 89770.113 14.780 0.3 212.9 14.3 3.0 3.8 1.5 Smallest isometric cube ROI H116.5% 144 1.63 577 5 94 892 45.084 7.457 1.8 134.3 18.1 3.0 3.6 1.6 H224.9% 152 1.88 471 8 194 870 45.084 11.219 0.8 180.5 16.1 4.0 5.3 2.3 H326.6% 173 1.82 475 7 221 908 45.084 11.993 0.9 172.6 14.4 3.8 5.2 1.8 H435.3% 155 2.41 352 19 326 880 45.084 15.903 −0.1 243.7 15.2 5.4 8.4 1.7W1 20.4% 166 1.32 754 3 220 902 45.084 9.178 0.4 134.6 14.6 3.0 3.7 1.5

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed, which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A method for producing a bone graft, comprisingthe steps of providing at least two bone matrix scaffolds, each bonematrix scaffold fabricated having at least one sheet exposed tomicrogravity over its development, wherein said at least one sheetmaterial forms a macrostructure developed under microgravity having asubstantially continuous network and voids with enhanced tensile loadingwhen exposed to normal gravitational forces wherein bone cell attachmentunder normal gravitational forces, loading, and integration will allrespond to a dynamic tension of subsequent compressive loading becauseeach of the at least two bone matrix scaffolds form an engineered staticstructure having at least one sheet exposed during development tomicrogravity and inherent porosity of the scaffold to facilitate bonematrix enhancement; connecting said at least two bone matrix scaffoldsusing fibers to produce the bone graft without cells; and depositing ofosseous material and/or chondral material with or without cells on saidbone graft, wherein the engineered static structure has enhancedmaterial properties that are stimulated by the tensile loading undernormal gravitational forces.
 2. The method according to claim 1, whereinsaid fibers comprise at least one polymer and/or a mixture of polymers.3. The method according to claim 2, wherein said at least one polymer isa copolymer having carboxylic acid groups and/or amine groups.
 4. Themethod according to claim 2, wherein said at least one polymer is aconductive polymer selected from polypyrrole, polyaniline,polyacetylene, polythiophene, polyurethane as a bioresorbable, netneutral charge material and mixtures thereof.
 5. The method according toclaim 1, wherein said fibers are collagen fibers.
 6. The methodaccording to claim 1, wherein said fibers are non-aldehyde cross-linkedtype I collagen.
 7. The method according to claim 5, wherein saidcollagen is chemically cross-linked with nordihydroguaiaretic acid. 8.The method according to claim 1, wherein said depositing of osseousand/or chondrial material on the bone graft includes incubating thedeposited osseous and/or chondrial material with cells for apredetermined period of time.
 9. The method according to claim 8,wherein said cells are selected from the group of living cells andrecombinant cells, chondrocytes, growth factor producing cells includingTGF-producing cells, and osteoblasts.
 10. The method according to claim8, further comprising removing said cells.
 11. The method according toclaim 1, further comprising shaping the graft.
 12. The method accordingto claim 11, wherein the shaping is performed by a method comprisingthree dimensional printing, cutting, moulding and/or pressing.
 13. Themethod according to claim 11, wherein the graft is shaped in the form ofa facial and/or cosmetic surgery graft.
 14. The method according toclaim 13, wherein the graft is shaped in a patient specific form. 15.The method according to claim 1, further comprising providingosteoinductive substances including proteins, to said graft.
 16. Themethod according to claim 15, wherein said osteoinductive substances arebone specific growth factors.
 17. The method of claim 1 comprising thesteps of; surgical, plastic and/or cosmetic bone replacement for apatient, comprising providing a graft and implanting said graft intosaid patient.
 18. The method according to claim 17, wherein saidimplanting is performed during cartilage, bone, joint, and/or articularcartilage repair and/or replacement.
 19. The method according to claim17, wherein said implanting comprises the direct implantation into thematrix of the bone to be replaced.
 20. The method according to claim 17,wherein said providing of said graft further comprises shaping thegraft.
 21. The method according to claim 20, wherein said shaping isperformed by a method comprising printing, cutting, moulding and/orpressing.
 22. The method according to claim 20, wherein said graft isshaped in the form of a facial and cosmetic surgery graft.
 23. Themethod according to claim 20, wherein said graft is shaped in apatient-specific form.